The World Health Organization reports that each year, more than 15% of women worldwide experience difficulties getting pregnant and seek medical assistance (WHO 1997), estimated to be 60 to 80 million women around the world a decade ago. Infertility is generally defined by the World Health Organization as a lack of conception after an arbitrary period of twelve months. However, many couples attempt for years to conceive naturally before seeking medical assistance in an effort to become pregnant. A decrease in fertility rate is associated with medical and non-medical factors. For example, women's age has been shown to be a direct major determinant of the average time required to conceive. It has been shown that premature ovarian failure occurs in 1:1000 women before the age of 30; 1:250 women by 35 years; and 1:100 by the age of 40. Therefore, the highest birth rates are in the age group of 25-30 years and declines sharply after the age of 35 years. Infertility is currently one of the most frequent health concerns facing the population aged 25-45 years. Thus, great interest and need exist for a method of extending fertility in healthy women, possibly taking away age-related barriers to childbearing, and for women who are unable to conceive through natural methods. Although infertility itself may not threaten physical health, it often has a serious impact on the emotional, mental and spiritual well-being of women and of couples.
Assisted reproductive technologies are procedures that involve extracorporeal handling of both human eggs (oocytes or ova) and sperm (spermatozoa), and of embryos for the purpose of establishing a pregnancy in a female subject. These procedures include, but are not limited to, in vitro fertilization (“IVF”) including embryo transfer, gamete intrafallopian transfer, zygote intrafallopian transfer, tubal embryo transfer, gamete and embryo cryopreservation, oocyte and embryo donation, and gestational surrogacy. In vitro fertilization (“IVF”) has evolved as the major treatment for infertility or sub-fertility when other methods of assisted reproductive technology have failed. In its most basic sense, the process involves extracting the female egg from a woman and fertilizing the egg by sperm outside the body (“in vitro”). The process involves monitoring a woman's ovulatory process, removing multiple eggs from the woman's ovaries and letting sperm fertilize the eggs in a fluid medium in a laboratory. The eggs are usually retrieved from the patient by transvaginal oocyte retrieval involving an ultrasound-guided needle piercing the vaginal wall to reach the ovaries. Through this needle, follicles can be aspirated, and the follicular fluid is handed to the IVF laboratory to identify and diagnose the ova. It is common to remove between ten and thirty eggs from each patient. The fertilized egg, (embryo), or usually multiple embryos, are then transferred to the patient's uterus with the intention of establishing a successful pregnancy. See, for example, U.S. Pat. No. 7,781,207.
First developed in the 1970's, in vitro fertilization has provided an effective form of assistance for a large proportion of women to date. Currently, it is reported that IVF accounts for 1.3% of all live births in Europe [Nygren et al. 2001] and 1.7% of all live births in Australasia [Hurst et al. 2001.] In the United States, assisted reproductive technology IVF cycles started in 2006 resulted in 41,343 births (54,656 infants), which was slightly more than 1.0% of total US births that year. In 2010, Robert Edwards was awarded the Nobel Prize in Physiology or Medicine for the development of in vitro fertilization. The next step was the ability to freeze and subsequently thaw and transfer embryos, first pioneered by Carl Wood, which significantly improved the feasibility of IVF treatment.
Typical successful pregnancy rates obtained with previous in vitro fertilization techniques remain relatively low, in the range of 15% to 25%, based on a number of reported studies. It is widely accepted by health care professionals that the most significant limiting factor to fertilization is the failure of embryos to implant into the endometrium, the lining of the uterus. Blake et al. report that 80-85% of embryos fail to result in pregnancy following transfer into IVF patients resulting in an enormous wastage of embryos. Simon et al. demonstrated that in women undergoing IVF cycle, implantation was detected in 60% of the cycles, therefore in all embryos transferred during IVF, 40% fail to implant.
One possible reason for low implantation rates during IVF is that embryos are transferred to the uterus two days after fertilization at the 4-8 cell stage. One view is that it may be more desirable to use embryos at the blastocyst stage reached at day 5-7 of culture. The advantages suggested include improved synchronization between embryo and uterus and the ability to select better quality embryos over the longer culture period. Blastocyst transfer may also help reduce the number of multiple births resulting from IVF by allowing the selection of fewer numbers of highly competent embryos per transfer. Typically during IVF procedure, two to five embryos are transferred to the uterus in order to increase the chance of implantation, creating the risk of multiple pregnancies. Consequently, more than half of all babies born in the United States after IVF treatment result from multiple gestations. It is sometimes the case that multiple implantations occur in the uterus and as the embryos continue to grow and develop within the womb, the patient is unable to carry more than one or two fetuses to term. At that point, a difficult decision is made to terminate those pregnancies and withdraw the additional fetus or fetuses from the womb, a process called embryo or fetus reduction, a procedure to reduce the number of viable embryos or fetuses in a multiple pregnancy. Such decisions carry with them both ethical and often psychologically traumatic implications. Development of laboratory techniques which would increase the probability and certainty of implantation of each embryo are still desired for a number of the aforementioned reasons.
In a process called natural cycle in vitro fertilization, the fertilization is performed by collecting one or more naturally selected eggs from the patient during a woman's natural menstrual cycle without the use of any drugs. In modified natural cycle IVF, fertility medication is used for two to four days during a woman's natural cycle to avoid spontaneous ovulation and to make the treatment more successful. “Mild IVF” is a method where a small dose of ovarian stimulating drugs are used for a short duration during a woman's natural cycle aimed at producing 2-7 eggs and creating healthy embryos. This method appears to reduce complications and side-effects for women and it is aimed at quality, and not quantity of eggs and embryos. However, this method yields a very low success rate of pregnancy.
Additional techniques are therefore used routinely in order to increase the chance of pregnancy. The most common is ovarian hyperstimulation or super-ovulation that is used in order to stimulate the ovaries to produce multiple eggs that are then retrieved from the patient. The long protocol typically involves downregulation (suppression or exhaustion) of the pituitary ovarian axis by the prolonged use of a gonadotropin releasing hormone (GnRH) agonist. Subsequent ovarian hyperstimulation, typically using follicle stimulating hormone (FSH), starts once the process of downregulation is complete, generally after 10 to 14 days. An IVF cycle using this protocol is known as conventional in vitro fertilization. The short protocol skips the downregulation procedure, and consists of a regimen of fertility medications to stimulate the development of multiple follicles of the ovaries. Other procedures use gonadotrophin-releasing hormone agonists (GnRHA), which decreases the need for monitoring by preventing premature ovulation, and more recently gonadotrophin-releasing hormone antagonists (GnRH Ant) have been used, which have a similar function. In most patients, injectable gonadotropins (usually FSH analogues) are used under close monitoring. Such monitoring frequently checks the estradiol level of the patient and, by means of gynecologic ultrasonography, follicular growth. Typically, approximately 10 days of injections are necessary. Ovarian stimulation carries the risk of excessive stimulation leading to ovarian hyperstimulation syndrome (OHSS), a potentially life-threatening complication of abdominal distension, ovarian enlargement, and respiratory, hemodynmic and metabolic complications. In addition, it has also of recent been demonstrated that fertility drugs used for the stimulation of ovulation of patients undergoing IVF treatment contribute to compromised implantation receptivity of the embryo in the uterus and lead to a decreased rate of pregnancy inception. [Ertzeid et al. 2001].
Female fertility can be affected by dysfunctions of the reproductive tract, of the neuroendocrine system or of the immune system. Some female cancer patients risk losing their fertility because certain kinds of chemotherapy treatments, and certain types of radiation treatments, can bring on premature menopause rendering them sterile. In Western Europe and North America, endocrine dysfunction is identified in about 10 to 20% of women presenting with infertility [Crosignani et al. 2000] Still, in about 10 to 20% of cases, the cause of infertility remains unknown. It is postulated that autoimmune reactions of the body may be the cause of infertility in many such women. Reproductive autoimmune failure and defects can be associated with overall activation of the immune system or with immune system reactions that are specifically directed against ovarian antigens.
Haller-Kikkatalo explains that active tolerance mechanisms are required to prevent self-protective inflammatory responses in the human body to the many foreign air-borne and food antigens that are encountered at the body's mucosal surfaces. However, the most important aspect of tolerance is self-tolerance, which prevents the body from mounting and immune attack against its own tissues—this is the prevention from autoimmune reactions. Autoimmunity is associated with an imbalance of various components of immune response and with the development of autoantibodies directed against normal host antigens. Female fertility is regulated by a series of highly coordinated and synchronized interactions in the hypothalamic-pituitary-ovarian axis. Reproductive autoimmune failure syndrome was originally described by Gleicher et. al. in women with endometriosis, infertility and increased autoantibodies. Although the impact of particular autoantibodies on the pathogenesis of infertility is not yet uniformly understood, autoimmune mechanisms as well as an increased production of multiple autoantibodies are involved in such infertility disorders as premature ovarian failure (POF), subclinical ovarian failure, recurrent pregnancy loss, endometriosis, polycystic ovary syndrome (PCOS), unexplained infertility, repeatedly unsuccessful IVF attempts, and spontaneous abortions. Some studies have suggested the lesser importance of specific antibodies and stressed the key role of overall activation of the immune system in reduced fecundity. [N. Gleicher, 2001; Dmowski et al., 1995.]
A group of research has focused on developing approaches to overcome immune-related infertility. The first and most commonly used approach is the use of medications that aim to suppress the autoimmune response in the patient in order to allow inception of pregnancy. For example, low dose oral prednisolone therapy was suggested for improving pregnancy rate in patients with recurrent IVF failure. However, contradicting data exist indicating that certain antibodies damage the embryo, interfere with the implantation process or interfere with the formation of the placenta. This renders it difficult to predict the success of therapy using these particular immunosuppressive medications. Methods have developed using milder pretreatment with acetylsalicylic acid or heparin. However, though this treatment has become generally universal, the rate of pregnancy using this approach remains relatively low. [Maghraby et al., 2007]
A second approach has recently been introduced. The procedure involves the preparation both of the endometrium and its surrounding environment by means of peripheral blood mononuclear cells (PBMCs). [Fujiwara et al., Kosaka et al., Yoshioka et al.] Peripheral blood mononuclear cells (PBMCs) allow the endometrium of the uterus to grow in sufficient size prior to embryo implantation and also act as building blocks by the body for growing the embryo(s) after their implantation in the uterus of the woman. According to proponents, PBMCs have been identified as multipotent cells. Multipotent cells produce cells of a particular lineage or closely related family. They have been shown to have the capability to be naturally transformed into any kind of human tissue. Multipotent cells are a valuable resource for research and therapeutic treatments. Recent advances in bioengineering are quite promising in repairing, building, engineering, regenerating, generating and growing tissue.
European Pat. Application EP1581637 discloses monocyte derived adult stem cells that are isolated from peripheral blood of mammals and methods of preparing, propagating and using these stem cells. The inventors of U.S. Pat. No. 7,795,018, M. Kuwana and H. Kodamo, disclose monocyte-derived multipotent cells (MOMCs) that can differentiate into endothelial cells by a medium culture under conditions inducing differentiation into endothelium. Further disclosed is a method for preparing MOMCs involving culturing PBMCs in vitro on fibronectin and collecting fibroblast-like cells. It was demonstrated that by culturing in a EBM-2 medium, a maintenance medium of endothelial cells, for seven days, MOMCs differentiate into endothelial cells changing the cell's morphology from a spindle shape to a morphology having multiple projections (see U.S. Pat. No. 7,795,018). The researchers of the present invention hypothesized that the use of PBMCs can therefore be instrumental during the reproductive process and have incorporated the use of PBMCs into a novel technique of in vitro fertilization provided herein.
A group of various inception agents has been developed, which separately or in combination, can be applied during IVF treatment in order to promote the acceptance of the embryo by a woman's immune system. Among these agents, soluble human leukocyte antigen G (sHLA-G) appears to be promising. The s-HLA class of molecules has been recognized to be involved in immune response and in the modulation of the maternal-fetal immune relationship during pregnancy. Sher et al. in U.S. patent application Ser. No. 10/829,081 discloses having isolated sHLA-G from the culture media surrounding pooled developing embryos and blastocysts. They observed that the absence of sHLA-G in the supernatant surrounding groups of embryos in culture is associated with significantly reduced IVF implantation and pregnancy rates. They proposed that addition of sHLA-G to the medium in which embryos are cultured and/or delivered into the uterine environment through embryo transfer, would enhance implantation and pregnancy potential of those embryos. Though the technique is useful, neither this approach, nor any other alone has solved the problem of autoimmune infertility. It is suggested herein that such inception agents be used in conjunction with the procedure of the invention herein to improve the probability and success of pregnancy inception.
Finally, engineered glycolipid-like molecular constructs have been shown to be capable of modifying the embryos and enhancing the interaction between the embryo and the target tissue, the endometrium. Building of a macro-molecular matrix on the external surface of an embryo and loading of said matrix with certain agents for stimulation of pregnancy inception are the techniques that, according to proponents, will have very broad applications in IVF treatment in the future. Not only has the modification of embryos by this constructive approach been successfully demonstrated in an in-vitro culture system, but animals have given birth to healthy offspring derived from such modified embryos. However, this approach has not yet been tested in clinical trials and remains premature to be considered among current medical tools.
Much research has been directed to procedures for improving the probability of successful pregnancy and birth of a child. Despite the considerable research, technical advances and variations in procedures, the rate of successful pregnancy utilizing IVF treatment still remains on average in the order of 15-25% per cycle. The researchers herein have attempted to address the challenge of embryo implantation and decrease the risk of autoimmune system responses by presenting a solution focusing on stabilizing the interaction between a woman's immune system and the embryo.